Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The invention relates to antibody molecules having specificity for
antigenic determinants of human IL-13, therapeutic uses of the antibody
molecules and methods for producing said antibody molecules.

Claims:

1. An antagonistic antibody which binds human IL-13 comprising a heavy
chain, wherein the variable domain of the heavy chain comprises at least
one of a CDR having the sequence given in SEQ ID NO:1 for CDR-H1, a CDR
having the sequence given in SEQ ID NO:2 for CDR-H2 and a CDR having the
sequence given in SEQ ID NO:3 for CDR-H3.

2. An antagonistic antibody according to claim 1, wherein the variable
domain of the heavy chain comprises the sequence given in SEQ ID NO:1 for
CDR-H1, the sequence given in SEQ ID NO:2 for CDR-H2 and the sequence
given in SEQ ID NO:3 for CDR-H3.

3. An antagonistic antibody which binds human IL-13, comprising a light
chain, wherein the variable domain of the light chain comprises at least
one of a CDR having the sequence given in SEQ ID NO:4 for CDR-L1, a CDR
having the sequence given in SEQ ID NO:5 for CDR-L2 and a CDR having the
sequence given in SEQ ID NO:6 for CDR-L3.

4. An antagonistic antibody according to claim 1 or claim 2, additionally
comprising a light chain, wherein the variable domain of the light chain
comprises at least one of a CDR having the sequence given in SEQ ID NO:4
for CDR-L1, a CDR having the sequence given in SEQ ID NO:5 for CDR-L2 and
a CDR having the sequence given in SEQ ID NO:6 for CDR-L3.

5. An antagonistic antibody according to claim 3 or claim 4 wherein the
variable domain of the light chain comprises the sequence given in SEQ ID
NO:4 for CDR-L1, the sequence given in SEQ ID NO:5 for CDR-L2 and the
sequence given in SEQ ID NO:6 for CDR-L3.

6. An antagonistic antibody which binds human IL-13, wherein the variable
domain of the heavy chain comprises three CDRs and the sequence of CDRH-1
has at least 60% identity or similarity to the sequence given in SEQ ID
NO:1, the sequence of CDRH-2 has at least 60% identity or similarity to
the sequence given in SEQ ID NO:2 and the sequence of CDRH-3 has at least
60% identity or similarity to the sequence given in SEQ ID NO:3.

7. An antagonistic antibody according to claim 6, additionally comprising
a light chain, wherein the variable domain of the light chain comprises
three CDRs and the sequence of CDRL-1 has at least 60% identity or
similarity to the sequence given in SEQ ID NO:4, the sequence of CDRL-2
has at least 60% identity or similarity to the sequence given in SEQ ID
NO:5 and the sequence of CDRL-3 has at least 60% identity or similarity
to the sequence given in SEQ ID NO:6.

8. An antibody according to any one of claims 1 to 5 wherein the heavy
chain comprises the sequence given in SEQ ID NO:31.

9. An antibody according to any one of claims 1 to 5, wherein the light
chain comprises the sequence given in SEQ ID NO:23.

10. A neutralising antibody molecule according to any one of claims 1 to
9, wherein the antibody molecule is selected from the group consisting
of: a complete antibody molecule having full length heavy and light
chains or a fragment thereof, such as a Fab, modified Fab', Fab',
F(ab')2, Fv, VH, VL or scFv fragment.

11. An antagonistic antibody which binds human IL-13, having a heavy
chain comprising the sequence given in SEQ ID NO:31 and a light chain
comprising the sequence given in SEQ ID NO:23.

12. An antagonistic antibody which binds human IL-13, wherein the
variable domain of the light chain comprises a sequence having at least
80% identity or similarity to the light chain variable domain of the
antibody of claim 11 and wherein the variable domain of the heavy chain
comprises a sequence having at least 80% identity or similarity to the
heavy chain variable domain of the antibody of claim 11.

13. An antagonistic antibody which binds human IL-13, having a heavy
chain comprising the sequence given in SEQ ID NO:35 and a light chain
comprising the sequence given in SEQ ID NO:27.

14. A neutralising antibody which binds human IL-13, in which the heavy
and light chains are at least 80% identical or similar to the
corresponding heavy and light chains of the antibody of claim 13.

15. A neutralising antibody molecule according to any one of claims 1 to
14, having an effector or a reporter molecule attached to it.

16. An antagonistic antibody having a binding affinity for isolated human
IL-13 of 30 pM or better.

17. An isolated DNA sequence encoding the heavy and/or light chain(s) of
an antibody according to any one of claims 1 to 16.

18. A cloning or expression vector comprising one or more DNA sequences
according to claim 17.

19. A vector according to claim 18, wherein the vector comprises the
sequences given in SEQ ID NO:36 and SEQ ID NO:28.

20. A host cell comprising one or more cloning or expression vectors
according to claim 19.

21. A process for the production of the antibody of any one of claims 1
to 16, comprising culturing the host cell of claim 20 and isolating the
antibody.

22. A pharmaceutical composition comprising an antibody according to any
one of claims 1 to 19, in combination with one or more of a
pharmaceutically acceptable excipient, diluent or carrier.

23. A pharmaceutical composition according to claim 22, additionally
comprising other active ingredients.

24. An antibody according to any one of claims 1 to 16 or a
pharmaceutical composition according to claim 22 or claim 23, for use in
the treatment or prophylaxis of a pathological disorder that is mediated
by IL-13 or that is associated with an increased level of IL-13.

25. The use of an antibody according to any one of claims 1 to 16 in the
manufacture of a medicament for the treatment or prophylaxis of a
pathological disorder that is mediated by IL-13 or that is associated
with an increased level of IL-13.

26. A method for the treatment of a human subject suffering from or at
risk of a pathological disorder mediated by IL-13, the method comprising
administering to the subject an effective amount of an anti-IL-13
antibody Fab or Fab' fragment by inhalation.

27. The method according to claim 26 wherein the antibody Fab or Fab'
fragment is an antibody according to any one of claims 1 to 16.

Description:

[0001] The present invention relates to IL-13 antibodies and fragments
thereof such as binding fragments thereof, compositions comprising the
same, and specifically to their use in the prevention and/or treatment of
various diseases including asthma, allergy, COPD, fibrosis, and/or
cancer.

[0013] Efficacy of anti-IL-13 treatment was also demonstrated in a chronic
model of murine asthma. In addition to exhibiting features of mucus
hyper-secretion and AHR, this model of chronic asthma demonstrates
several hallmarks of human disease that are lacking in the more acute
models. These include eosinophilia of the lung tissue located in
inter-epithelial spaces as well as smooth muscle fibrosis as measured by
increases in collagen deposition. The chronic asthma model is induced
with repeated aerosol challenges with OVA in OVA-sensitized mice
1×/week for a total of 4 weeks. Anti-IL-13 antibody administered
for the final 2 weeks of OVA challenges (from day 36 with efficacy
readouts assessed on day 53 of study) significantly inhibited AHR,
pulmonary inflammation, goblet cell hyperplasia, mucus hypersecretion,
and airway fibrosis (Yang, Li et al. 2005 J Pharmacol Exp Ther).

[0014] The therapeutic effect of IL-13 antagonist was also demonstrated to
inhibit AHR in a primate model of asthma, (American Thoracic Society, San
Diego 2005).

[0016] Due to the role of human IL-13 in a variety of human disorders,
therapeutic strategies have been designed to inhibit or counteract IL-13
activity. In particular, antibodies that bind to, and neutralize, IL-13
have been sought as a means to inhibit IL-13 activity. However, there
exists a need in the art for suitable and/or improved antibodies capable
of binding IL-13, especially human IL-13. In particular the antibodies
are capable of neutralizing human IL-13. The present invention provides a
novel family of binding proteins, CDR grafted antibodies, humanized
antibodies, and fragments thereof, capable of binding human IL-13,
binding with high affinity, and binding and neutralizing human IL-13.

[0064] shows an alignment of the light chains for the
rat, acceptor framework and the humanised light chains and also heavy
chains. CDRs are in bold and underlined. Donor residues G49 and R71 are
in bold, italic and highlighted.

[0066] FIG. 14. Effect of Ab652 on the BAL eosinophil count measured 24 h
after allergen challenge. Data are normalised to the BAL eosinophil count
measured in the screening phase of the study. Mean±SEM, n=4-8 per
group.

[0067] FIG. 15. Effect of Ab652 on peak airway resistance measured up to
15 minutes after allergen challenge. Data are expressed as mean±SEM,
n=4-8 per group.

[0069] The residues in antibody variable domains are conventionally
numbered according to a system devised by Kabat et al. This system is set
forth in Kabat et al., 1987, in Sequences of Proteins of Immunological
Interest, US Department of Health and Human Services, NIH, USA (hereafter
"Kabat et al. (supra)"). This numbering system is used in the present
specification except where otherwise indicated.

[0070] The Kabat residue designations do not always correspond directly
with the linear numbering of the amino acid residues. The actual linear
amino acid sequence may contain fewer or additional amino acids than in
the strict Kabat numbering corresponding to a shortening of, or insertion
into, a structural component, whether framework or complementarity
determining region (CDR), of the basic variable domain structure. The
correct Kabat numbering of residues may be determined for a given
antibody by alignment of residues of homology in the sequence of the
antibody with a "standard" Kabat numbered sequence.

[0071] The CDRs of the heavy chain variable domain are located at residues
31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3)
according to the Kabat numbering system. However, according to Chothia
(Chothia, C. and Lesk, A.M. J. Mol. Biol., 196, 901-917 (1987)), the loop
equivalent to CDR-H1 extends from residue 26 to residue 32. Thus
`CDR-H1`, as used herein, comprises residues 26 to 35, as described by a
combination of the Kabat numbering system and Chothia's topological loop
definition.

[0072] The CDRs of the light chain variable domain are located at residues
24 to 34 CDR-L1), residues 50 to 56 (CDR-L2) and residues 89 to 97
(CDR-L3) according to the Kabat numbering system.

[0073] In one embodiment the antibody is an antagonistic antibody.

[0074] As used herein, the term `antagonistic antibody` describes an
antibody that is capable of inhibiting and/or neutralising the biological
signalling activity of IL-13, for example by blocking binding or
substantially reducing binding of IL-13 to IL-13 receptor and thus
inhibiting the activation of the receptor.

[0075] Antibodies for use in the present invention may be obtained using
any suitable method known in the art. The IL-13 polypeptide, including a
fusion polypeptide containing IL-13, or cells (recombinantly) expressing
the polypeptide can be used to produce antibodies which specifically
recognise IL-13. The IL-13 polypeptide may be the `mature` polypeptide or
a biologically active fragment or derivative thereof. IL-13 polypeptides
may be prepared by processes well known in the art from genetically
engineered host cells comprising expression systems or they may be
recovered from natural biological sources. In the present application,
the term "polypeptides" includes peptides, polypeptides and proteins.
These are used interchangeably unless otherwise specified. The IL-13
polypeptide may in some instances be part of a larger protein such as a
fusion protein for example fused to an affinity tag.

[0076] Antibodies generated against the IL-13 polypeptide may be obtained,
where immunisation of an animal is necessary, by administering the
polypeptides to an animal, preferably a non-human animal, using
well-known and routine protocols, see for example Handbook of
Experimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell Scientific
Publishers, Oxford, England, 1986). Many warm-blooded animals, such as
rabbits, mice, rats, sheep, cows, camels or pigs may be immunized.
However, mice, rabbits, pigs and rats are generally most suitable.

[0077] Antibodies for use in the present invention include whole
antibodies and functionally active fragments or derivatives thereof and
may be, but are not limited to, monoclonal, humanised, fully human or
chimeric antibodies.

[0079] Antibodies for use in the invention may also be generated using
single lymphocyte antibody methods by cloning and expressing
immunoglobulin variable region cDNAs generated from single lymphocytes
selected for the production of specific antibodies by for example the
methods described by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA
93(15):7843-78481; WO92/02551; WO2004/051268 and International Patent
Application number WO2004/106377.

[0080] Screening for antibodies can be performed using assays to measure
binding to IL-13 and/or assays to measure the ability to block the
binding of IL-13 to one or more of it's receptors. An example of a
binding assay is an ELISA, for example, using a fusion protein of IL-13,
which is immobilized on plates, and employing a conjungated secondary
antibody to detect anti-IL-13 antibody bound to the IL-13. An example of
a blocking assay is a flow cytometry based assay measuring the blocking
of IL-13 ligand protein binding to an IL-13R. A fluorescently labelled
secondary antibody is used to detect the amount of IL-13 ligand protein
binding to the IL-13R.

[0081] Humanised antibodies (which include CDR-grafted antibodies) are
antibody molecules having one or more complementarity determining regions
(CDRs) from a non-human species and a framework region from a human
immunoglobulin molecule (see, e.g. U.S. Pat. No. 5,585,089; WO91/09967).
It will be appreciated that it may only be necessary to transfer the
specificity determining residues of the CDRs rather than the entire CDR
(see for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanised
antibodies may optionally further comprise one or more framework residues
derived from the non-human species from which the CDRs were derived.

[0082] Chimeric antibodies are composed of elements derived from two
different species such that the element retains the characteristics of
the species from which it is derived. Generally a chimeric antibody will
comprise a variable region from one species, for example a mouse, rat,
rabbit or similar and constant region from another species such as a
human.

[0084] Fully human antibodies are those antibodies in which the variable
regions and the constant regions (where present) of both the heavy and
the light chains are all of human origin, or substantially identical to
sequences of human origin, but not necessarily from the same antibody.
Examples of fully human antibodies may include antibodies produced, for
example by the phage display methods described above and antibodies
produced by mice in which the murine immunoglobulin variable and
optionally the constant region genes have been replaced by their human
counterparts eg. as described in general terms in EP 0546073, U.S. Pat.
No. 5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126, U.S.
Pat. No. 5,633,425, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,770,429, EP
0438474 and EP 0463151.

[0085] In one embodiment the present invention provides an antagonistic
antibody having specificity for human IL-13, comprising a heavy chain,
wherein the variable domain of the heavy chain comprises at least one CDR
having the sequence given in FIG. 1, SEQ ID NO:1 for CDR-H1, a CDR having
the sequence given in SEQ ID NO:2 for CDR-H2 and/or a CDR having the
sequence given in SEQ ID NO:3 for CDR-H3.

[0086] In another embodiment the present invention provides an
antagonistic antibody having specificity for human IL-13, comprising a
heavy chain, wherein at least two of CDR-H1, CDR-H2 and CDR-H3 of the
variable domain of the heavy chain are selected from the following: the
sequence given in SEQ ID NO:1 for CDR-H1, the sequence given in SEQ ID
NO:2 for CDR-H2 and the sequence given in SEQ ID NO:3 for CDR-H3. For
example, the antibody may comprise a heavy chain wherein CDR-H1 has the
sequence given in SEQ ID NO:1 and CDR-H2 has the sequence given in SEQ ID
NO:2. Alternatively, the antibody may comprise a heavy chain wherein
CDR-H1 has the sequence given in SEQ ID NO:1 and CDR-H3 has the sequence
given in SEQ ID NO:3, or the antibody may comprise a heavy chain wherein
CDR-H2 has the sequence given in SEQ ID NO:2 and CDR-H3 has the sequence
given in SEQ ID NO:3. For the avoidance of doubt, it is understood that
all permutations are included.

[0087] In another embodiment the present invention provides an
antagonistic antibody having specificity for human IL-13, comprising a
heavy chain, wherein the variable domain of the heavy chain comprises the
sequence given in SEQ ID NO:1 for CDR-H1, the sequence given in SEQ ID
NO:2 for CDR-H2 and the sequence given in SEQ ID NO:3 for CDR-H3.

[0088] In one embodiment the present invention provides an antagonistic
antibody having specificity for human IL-13, comprising a light chain,
wherein the variable domain of the light chain comprises at least one CDR
having the sequence given in FIG. 1, SEQ ID NO:4 for CDR-L1, a CDR having
the sequence given in SEQ ID NO:5 for CDR-L2 and/or a CDR having the
sequence given in SEQ ID NO:6 for CDR-L3.

[0089] In another embodiment the present invention provides an
antagonistic antibody having specificity for human IL-13, comprising a
light chain, wherein at least two of CDR-L1, CDR-L2 and CDR-L3 of the
variable domain of the light chain are selected from the following: the
sequence given in SEQ ID NO:4 for CDR-L1, the sequence given in SEQ ID
NO:5 for CDR-L2 and the sequence given in SEQ ID NO:6 for CDR-L3. For
example, the antibody may comprise a light chain wherein CDR-L1 has the
sequence given in SEQ ID NO:4 and CDR-L2 has the sequence given in SEQ ID
NO:5. Alternatively, the antibody may comprise a light chain wherein
CDR-L1 has the sequence given in SEQ ID NO:4 and CDR-L3 has the sequence
given in SEQ ID NO:6, or the antibody may comprise a light chain wherein
CDR-L2 has the sequence given in SEQ ID NO:5 and CDR-L3 has the sequence
given in SEQ ID NO:6. For the avoidance of doubt, it is understood that
all permutations are included.

[0090] In another embodiment the present invention provides an
antagonistic antibody having specificity for human IL-13, comprising a
light chain, wherein the variable domain comprises the sequence given in
SEQ ID NO:4 for CDR-L1, the sequence given in SEQ ID NO:5 for CDR-L2 and
the sequence given in SEQ ID NO:6 for CDR-L3.

[0091] The antibody molecules of the present invention suitably comprise a
complementary light chain or a complementary heavy chain, respectively.

[0092] Hence in one embodiment, an antibody according to the present
invention comprises a heavy chain, wherein the variable domain of the
heavy chain comprises the sequence given in SEQ ID NO:1 for CDR-H1, the
sequence given in SEQ ID NO:2 for CDR-H2 and/or the sequence given in SEQ
ID NO:3 for CDR-H3 and a light chain wherein the variable domain of the
light chain comprises the sequence given in SEQ ID NO:4 for CDR-L1, the
sequence given in SEQ ID NO:5 for CDR-L2 and/or the sequence given in SEQ
ID NO:6 for CDR-L3.

[0093] It will be appreciated that one or more amino acid substitutions,
additions and/or deletions may be made to the CDRs provided by the
present invention without significantly altering the ability of the
antibody to bind to IL-13 and to neutralise IL-13 activity. The effect of
any amino acid substitutions, additions and/or deletions can be readily
tested by one skilled in the art, for example by using the methods
described herein, particularly those illustrated in the Examples, to
determine IL-13 binding and inhibition of the IL-13/IL-13 receptor
interaction.

[0094] Accordingly, the present invention provides an antibody having
specificity for human IL-13 comprising one or more CDRs selected from
CDRH-1 (SEQ ID NO:1), CDRH-2 (SEQ ID NO:2), CDRH-3 (SEQ ID NO:3), CDRL-1
(SEQ ID NO:4), CDRL-2 (SEQ ID NO:5) and CDRL-3 (SEQ ID NO:6) in which one
or more amino acids in one or more of the CDRs has been substituted with
another amino acid, for example a similar amino acid as defined herein
below.

[0095] In one embodiment, the present invention provides an antibody
having specificity for human IL-13 comprising CDRH-1 (SEQ ID NO:1),
CDRH-2 (SEQ ID NO:2 or SEQ ID NO:20), CDRH-3 (SEQ ID NO:3), CDRL-1 (SEQ
ID NO:4), CDRL-2 (SEQ ID NO:5) and CDRL-3 (SEQ ID NO:6), for example in
which one or more amino acids in one or more of the CDRs has been
substituted with another amino acid, such as a similar amino acid as
defined herein below.

[0096] In one embodiment, an antibody of the present invention comprises a
heavy chain, wherein the variable domain of the heavy chain comprises
three CDRs wherein the sequence of CDRH-1 has at least 60% identity or
similarity to the sequence given in SEQ ID NO:1, CDRH-2 has at least 60%
identity or similarity to the sequence given in SEQ ID NO:2 and/or CDRH-3
has at least 60% identity or similarity to the sequence given in SEQ ID
NO:3. In another embodiment, an antibody of the present invention
comprises a heavy chain, wherein the variable domain of the heavy chain
comprises three CDRs wherein the sequence of CDRH-1 has at least 70%,
80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ
ID NO:1, CDRH-2 has at least 70%, 80%, 90%, 95% or 98% identity or
similarity to the sequence given in SEQ ID NO:2 and/or CDRH-3 has at
least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence
given in SEQ ID NO:3.

[0097] "Identity", as used herein, indicates that at any particular
position in the aligned sequences, the amino acid residue is identical
between the sequences. "Similarity", as used herein, indicates that, at
any particular position in the aligned sequences, the amino acid residue
is of a similar type between the sequences. For example, leucine may be
substituted for isoleucine or valine. Other amino acids which can often
be substituted for one another include but are not limited to:

[0103] In another embodiment, an antibody of the present invention
comprises a light chain, wherein the variable domain of the light chain
comprises three CDRs wherein the sequence of CDRL-1 has at least 60%
identity or similarity to the sequence given in SEQ ID NO:4, CDRL-2 has
at least 60% identity or similarity to the sequence given in SEQ ID NO:5
and/or CDRL-3 has at least 60% identity or similarity to the sequence
given in SEQ ID NO:6. In another embodiment, an antibody of the present
invention comprises a light chain, wherein the variable domain of the
light chain comprises three CDRs wherein the sequence of CDRL-1 has at
least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence
given in SEQ ID NO:4, CDRL-2 has at least 70%, 80%, 90%, 95% or 98%
identity or similarity to the sequence given in SEQ ID NO:5 and/or CDRL-3
has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the
sequence given in SEQ ID NO:6.

[0104] In one embodiment the antibody provided by the present invention is
a monoclonal antibody.

[0105] In one embodiment the antibody provided by the present invention is
a chimeric antibody.

[0106] In one embodiment the antibody provided by the present invention is
a CDR-grafted antibody molecule comprising one or more of the CDRs
provided in SEQ ID NOS:1, 2, 3, 4, 5, 6 or variants thereof. As used
herein, the term `CDR-grafted antibody molecule` refers to an antibody
molecule wherein the heavy and/or light chain contains one or more CDRs
(including, if desired, one or more modified CDRs) from a donor antibody
(e.g. a murine or rat monoclonal antibody) grafted into a heavy and/or
light chain variable region framework of an acceptor antibody (e.g. a
human antibody). For a review, see Vaughan et al, Nature Biotechnology,
16, 535-539, 1998. In one embodiment rather than the entire CDR being
transferred, only one or more of the specificity determining residues
from any one of the CDRs described herein above are transferred to the
human antibody framework (see for example, Kashmiri et al., 2005,
Methods, 36, 25-34). In one embodiment only the specificity determining
residues from one or more of the CDRs described herein above are
transferred to the human antibody framework. In another embodiment only
the specificity determining residues from each of the CDRs described
herein above are transferred to the human antibody framework.

[0107] When the CDRs or specificity determining residues are grafted, any
appropriate acceptor variable region framework sequence may be used
having regard to the class/type of the donor antibody from which the CDRs
are derived, including mouse, primate and human framework regions.
Suitably, the CDR-grafted antibody according to the present invention has
a variable domain comprising human acceptor framework regions as well as
one or more of the CDRs or specificity determining residues described
above. Thus, provided in one embodiment is a neutralising CDR-grafted
antibody wherein the variable domain comprises human acceptor framework
regions and non-human donor CDRs.

[0108] Examples of human frameworks which can be used in the present
invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al.,
supra). For example, KOL and NEWM can be used for the heavy chain, REI
can be used for the light chain and EU, LAY and POM can be used for both
the heavy chain and the light chain. Alternatively, human germline
sequences may be used; these are available at:
http://vbase.mrc-cpe.cam.ac.uk/

[0109] In a CDR-grafted antibody of the present invention, the acceptor
heavy and light chains do not necessarily need to be derived from the
same antibody and may, if desired, comprise composite chains having
framework regions derived from different chains.

[0110] The suitable framework region for the heavy chain of the
CDR-grafted antibody of the present invention is derived from the human
sub-group VH2 sequence 3-1 2-26 together with JH4 (SEQ ID NO:41).
Accordingly, provided is a neutralising CDR-grafted antibody comprising
at least one non-human donor CDR wherein the heavy chain framework region
is derived from the human subgroup VH2 sequence 3-1 2-26 together with
JH4. The sequence of human JH4 is as follows: (YFDY)WGQGTLVTVS (Seq ID
No: 43). The YFDY motif is part of CDR-H3 and is not part of framework 4
(Ravetch, J V. et al., 1981, Cell, 27, 583-591).

[0111] The suitable framework region for the light chain of the
CDR-grafted antibody of the present invention is derived from the human
germline sub-group VK1 sequence 2-1 1-02 together with JK4 (SEQ ID
NO:39). Accordingly, provided is a neutralising CDR-grafted antibody
comprising at least one non-human donor CDR wherein the light chain
framework region is derived from the human subgroup sequence 2-1 1-02
together with JK4. The JK4 sequence is as follows: (LT)FGGGTKVEIK (Seq ID
No: 44). The LT motif is part of CDR-L3 and is not part of framework 4
(Hieter, P A., et al., 1982, J. Biol. Chem., 257, 1516-1522).

[0112] In one embodiment a light and/or heavy framework is selected from a
sequence as shown in SEQ ID No: 39 to 42.

[0113] Also, in a CDR-grafted antibody of the present invention, the
framework regions need not have exactly the same sequence as those of the
acceptor antibody. For instance, unusual residues may be changed to more
frequently-occurring residues for that acceptor chain class or type.
Alternatively, selected residues in the acceptor framework regions may be
changed so that they correspond to the residue found at the same position
in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324).
Such changes should be kept to the minimum necessary to recover the
affinity of the donor antibody. A protocol for selecting residues in the
acceptor framework regions which may need to be changed is set forth in
WO 91/09967.

[0114] Suitably, in a CDR-grafted antibody molecule of the present
invention, if the acceptor heavy chain has the human VH2 sequence 3-12-26
together with JH4, then the acceptor framework regions of the heavy chain
comprise, in addition to one or more donor CDRs, a donor residue at at
least one of positions 49 and 71 (according to Kabat et al., (supra))
(See FIG. 12).

[0115] Accordingly, provided is a CDR-grafted antibody, wherein at least
the residues at positions 49 and 71 of the variable domain of the heavy
chain are donor residues.

[0116] Donor residues are residues from the donor antibody, i.e. the
antibody from which the CDRs were originally derived. Preferably the
residues are Glycine and Arginine at positions 49 and 71 respectively.

[0117] In one embodiment, an antibody of the present invention comprises a
heavy chain, wherein the variable domain of the heavy chain comprises the
sequence given in SEQ ID NO: 31

[0118] It will be appreciated that one or more amino acid substitutions,
additions and/or deletions may be made to the antibody variable domains,
provided by the present invention, without significantly altering the
ability of the antibody to bind to IL-13 and to neutralise IL-13
activity. The effect of any amino acid substitutions, additions and/or
deletions can be readily tested by one skilled in the art, for example by
using the methods described in the Examples to determine IL-13 binding
and/or ligand/receptor blocking.

[0119] In another embodiment, an antibody of the present invention
comprises a heavy chain, wherein the variable domain of the heavy chain
comprises a sequence having at least 60% identity or similarity to the
sequence given in SEQ ID NO:31. In one embodiment, an antibody of the
present invention comprises a heavy chain, wherein the variable domain of
the heavy chain comprises a sequence having at least 70%, 80%, 90%, 95%
or 98% identity or similarity to the sequence given in SEQ ID NO: 31.

[0120] In one embodiment, an antibody of the present invention comprises a
light chain, wherein the variable domain of the light chain comprises the
sequence given in SEQ ID NO: 23.

[0121] In another embodiment, an antibody of the present invention
comprises a light chain, wherein the variable domain of the light chain
comprises a sequence having at least 60% identity or similarity to the
sequence given in SEQ ID NO:23. In one embodiment the antibody of the
present invention comprises a light chain, wherein the variable domain of
the light chain comprises a sequence having at least 70%, 80%, 90%, 95%
or 98% identity or similarity to the sequence given in SEQ ID NO: 23.

[0122] In one embodiment an antibody of the present invention comprises a
heavy chain, wherein the variable domain of the heavy chain comprises the
sequence given in SEQ ID NO:31 and a light chain, wherein the variable
domain of the light chain comprises the sequence given in SEQ ID NO:23.

[0123] In another embodiment of the invention, the antibody comprises a
heavy chain and a light chain, wherein the variable domain of the heavy
chain comprises a sequence having at least 60% identity or similarity to
the sequence given in SEQ ID NO:31 and the variable domain of the light
chain comprises a sequence having at least 60% identity or similarity to
the sequence given in SEQ ID NO:23. Suitably, the antibody comprises a
heavy chain, wherein the variable domain of the heavy chain comprises a
sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity
to the sequence given in SEQ ID NO:31 and a light chain, wherein the
variable domain of the light chain comprises a sequence having at least
70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in
SEQ ID NO:23.

[0124] The antibody molecules of the present invention may comprise a
complete antibody molecule having full length heavy and light chains or a
fragment thereof and may be, but are not limited to Fab, modified Fab,
Fab', modified Fab', F(ab')2, Fv, single domain antibodies (e.g. VH
or VL or VHH), scFv, bi, tri or tetra-valent antibodies, Bis-scFv,
diabodies, triabodies, tetrabodies and epitope-binding fragments of any
of the above (see for example Holliger and Hudson, 2005, Nature Biotech.
23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews--Online
2(3), 209-217). The methods for creating and manufacturing these antibody
fragments are well known in the art (see for example Verma et al., 1998,
Journal of Immunological Methods, 216, 165-181). Other antibody fragments
for use in the present invention include the Fab and Fab' fragments
described in International patent applications WO 2005/003169, WO
2005/003170 and WO 2005/003171 and Fab-dAb fragments described in
International patent application WO2009/040562. Multi-valent antibodies
may comprise multiple specificities or may be monospecific (see for
example WO 92/22853 and WO 05/113605).

[0125] The constant region domains of the antibody molecule of the present
invention, if present, may be selected having regard to the proposed
function of the antibody molecule, and in particular the effector
functions which may be required. For example, the constant region domains
may be human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG
constant region domains may be used, especially of the IgG1 and IgG3
isotypes when the antibody molecule is intended for therapeutic uses and
antibody effector functions are required. Alternatively, IgG2 and IgG4
isotypes may be used when the antibody molecule is intended for
therapeutic purposes and antibody effector functions are not required,
e.g. for simply blocking IL-13 activity. It will be appreciated that
sequence variants of these constant region domains may also be used. For
example IgG4 molecules in which the serine at position 241 has been
changed to proline as described in Angal et al., Molecular Immunology,
1993, 30 (1), 105-108 may be used. It will also be understood by one
skilled in the art that antibodies may undergo a variety of
posttranslational modifications. The type and extent of these
modifications often depends on the host cell line used to express the
antibody as well as the culture conditions. Such modifications may
include variations in glycosylation, methionine oxidation,
diketopiperazine formation, aspartate isomerization and asparagine
deamidation. A frequent modification is the loss of a carboxy-terminal
basic residue (such as lysine or arginine) due to the action of
carboxypeptidases (as described in Harris, RJ. Journal of Chromatography
705:129-134, 1995). However, there is no C-terminal Lysine on either
heavy or light chain of Ab652 embodiment of the invention.

[0126] In one embodiment the antibody heavy chain comprises a CH1 domain
and the antibody light chain comprises a CL domain, either kappa or
lambda.

[0127] In one embodiment the antibody provided by the present invention is
an antagonistic antibody having specificity for human IL-13 in which the
heavy chain constant region comprises a modified hinge region.
Accordingly, the present invention provides an antibody in which the
heavy chain comprises or consists of the sequence given in SEQ ID No: 35

[0128] It will be appreciated that one or more amino acid substitutions,
additions and/or deletions may be made to the antibody variable and/or
constant domains provided by the present invention without significantly
altering the ability of the antibody to bind to IL-13 and to neutralise
IL-13 activity. The effect of any amino acid substitutions, additions
and/or deletions can be readily tested by one skilled in the art, for
example by using the methods described herein, particularly those
illustrated in the Examples, to determine IL-13 binding and blocking of
the IL-13/IL-13 receptor interaction.

[0129] In one embodiment of the invention, the antibody comprises a heavy
chain, wherein the heavy chain comprises a sequence having at least 60%
identity or similarity to the sequence given in SEQ ID NO: 35. Suitably,
the antibody comprises a heavy chain, wherein the heavy chain comprises a
sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity
to the sequence given in SEQ ID NO: 35.

[0130] In one embodiment an antibody molecule according to the present
invention comprises a light chain comprising the sequence given in SEQ ID
NO: 27.

[0131] In one embodiment of the invention, the antibody comprises a light
chain, wherein the light chain comprises a sequence having at least 60%
identity or similarity to the sequence given in SEQ ID NO: 27. For
example, the antibody comprises a light chain, wherein the light chain
comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity
or similarity to the sequence given in SEQ ID NO: 27.

[0132] In one embodiment the present invention provides an antibody in
which the heavy chain comprises or consists of the sequence given in SEQ
ID NO:35 and the light chain comprises or consists of the sequence given
in SEQ ID NO:27.

[0133] In one embodiment of the invention, the antibody comprises a heavy
chain and a light chain, wherein the heavy chain comprises a sequence
having at least 60% identity or similarity to the sequence given in SEQ
ID NO:35 and the light chain comprises a sequence having at least 60%
identity or similarity to the sequence given in SEQ ID NO:27. Generally,
the antibody comprises a heavy chain, wherein the heavy chain comprises a
sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity
to the sequence given in SEQ ID NO:35 and a light chain, wherein the
light chain comprises a sequence having at least 70%, 80%, 90%, 95% or
98% identity or similarity to the sequence given in SEQ ID NO:27.

[0134] Biological molecules, such as antibodies or fragments, contain
acidic and/or basic functional groups, thereby giving the molecule a net
positive or negative charge. The amount of overall "observed" charge will
depend on the absolute amino acid sequence of the entity, the local
environment of the charged groups in the 3D structure and the
environmental conditions of the molecule. The isoelectric point (pI) is
the pH at which a particular molecule or surface carries no net
electrical charge. In one embodiment the antibody or fragment according
to the present disclosure has an isoelectric point (pI) of at least 7. In
one embodiment the antibody or fragment has an isoelectric point of at
least 8, such as 8.5, 8.6, 8.7, 8.8 or 9. In one embodiment the pI of the
antibody is 8.

[0135] The IL-13 antibody and fragments of the invention may be engineered
to have an appropriate isoelectric point. This may lead to antibodies
and/or fragments with more robust properties, in particular suitable
solubility and/or stability profiles.

[0136] Thus in one aspect the invention provides a humanised IL-13
antibody engineered to have an isoelectric point different to that of the
originally identified antibody. The antibody may, for example be
engineered by replacing an amino acid residue such as replacing an acidic
amino acid residue with one or more basic amino acid residues.
Alternatively, basic amino acid residues may be added or acidic amino
acid residues can be removed. Alternatively, if the molecule has an
unacceptably high pI value acidic residues may be introduced to lower the
pH, as required. The pI of the engineered antibody or fragment may, for
example be 8 or above, such 8.5 or 9. It is important that when
manipulating the pI, then care must be taken to retain the desirable
activity of the antibody or fragment. Thus in one embodiment the
engineered antibody or fragment has the same or substantially the same
activity as the "unmodified" antibody or fragment.

[0137] Programs such as **ExPASY http://www.expasy.ch/tools/pi_tool.html,
and

[0138] http://www.iut-arles.up.univ-mrs.fr/w3bb/d_abim/compo-p.html, may
be used to predict the isoelectric point of the antibody or fragment.

[0139] In one embodiment the antibodies of the present invention are
suitable for inhaled delivery, for example, by nebulisation. In one
example the physical properties of the antibodies of the present
invention e.g. binding affinity and potency are not substantially altered
by nebulisation. In one example the antibodies of the present invention
are highly stable. One measure of antibody stability is melting
temperature (Tm). Melting temperature may be determined by any suitable
method known in the art, for example using Thermofluor (Ericsson et al,
Analytical Biochemistry 357 (2006) 289-298) or DSC (differential scanning
calorimetry). Preferably the antibodies provided by the present invention
have a high melting temperature (Tm), typically of at least 75° C.
In one example the antibody of the present invention has a Tm of at least
75° C. In one example the antibody of the present invention has a
Tm of at least 80° C. In one example the antibody of the present
invention has a Tm of at least 83° C.

[0140] Also provided by the present invention is a specific region or
epitope of human IL-13 which is bound by an antibody provided by the
present invention, in particular an antibody comprising the heavy chain
sequence (SEQ ID NO:35) and/or the light chain sequence (SEQ ID NO:27).

[0141] This specific region or epitope of the human IL-13 polypeptide can
be identified by any suitable epitope mapping method known in the art in
combination with any one of the antibodies provided by the present
invention. Examples of such methods include screening peptides of varying
lengths derived from IL-13 for binding to the antibody of the present
invention with the smallest fragment that can specifically bind to the
antibody containing the sequence of the epitope recognised by the
antibody. The IL-13 peptides may be produced synthetically or by
proteolytic digestion of the IL-13 polypeptide. Peptides that bind the
antibody can be identified by, for example, mass spectrometric analysis.
In another example, NMR spectroscopy or X-ray crystallography can be used
to identify the epitope bound by an antibody of the present invention.
Once identified, the epitopic fragment which binds an antibody of the
present invention can be used, if required, as an immunogen to obtain
additional antagonistic antibodies which bind the same epitope.

[0142] Antibodies which cross-block the binding of an antibody according
to the present invention in particular, an antibody comprising the heavy
chain sequence (SEQ ID NO:31) and the light chain sequence (SEQ ID NO:27)
may be similarly useful in antagonising IL-13 activity. Accordingly, the
present invention also provides an antagonistic antibody having
specificity for human IL-13, which cross-blocks the binding of any one of
the antibodies described above to human IL-13 and/or is cross-blocked
from binding IL-13 by any one of those antibodies. In one embodiment,
such an antibody binds to the same epitope as an antibody described
herein above. In another embodiment the cross-blocking neutralising
antibody binds to an epitope which borders and/or overlaps with the
epitope bound by an antibody described herein above. In another
embodiment the cross-blocking neutralising antibody of this aspect of the
invention does not bind to the same epitope as an antibody of the present
invention or an epitope that borders and/or overlaps with said epitope.

[0143] Cross-blocking antibodies can be identified using any suitable
method in the art, for example by using competition ELISA or BIAcore
assays where binding of the cross blocking antibody to human IL-13
prevents the binding of an antibody of the present invention or vice
versa.

[0144] In one embodiment there is provided an antagonistic antibody having
specificity for human IL-13, which cross-blocks the binding of an
antibody whose heavy chain comprises the sequence shown in SEQ ID NO: 35
and whose light chain comprises the sequence shown in SEQ ID NO: 27 to
human IL-13. In one embodiment the cross-blocking antibodies provided by
the present invention inhibit the binding of an antibody comprising the
heavy chain sequence shown in SEQ ID NO:35 and the light chain sequence
shown in SEQ ID NO:27 by greater than 80%, for example by greater than
85%, such as by greater than 90%, in particular by greater than 95%.

[0145] Alternatively or in addition, antagonistic antibodies according to
this aspect of the invention may be cross-blocked from binding to human
IL-13 by an antibody comprising the heavy chain sequence shown in SEQ ID
NO:35 and the light chain sequence shown in SEQ ID NO: 27. Also provided
therefore is an antagonistic antibody molecule having specificity for
human IL-13 which is cross-blocked from binding human IL-13 by an
antibody comprising the heavy chain sequence shown in SEQ ID NO: 35 and
the light chain sequence shown in SEQ ID NO: 27. In one embodiment the
antagonistic antibodies provided by this aspect of the invention are
inhibited from binding human IL-13 by an antibody comprising the heavy
chain sequence shown in SEQ ID NO: 35 and the light chain sequence shown
in SEQ ID No: 27 by greater than 80%, for example by greater than 85%,
such as by greater than 90%, in particular by greater than 95%.

[0146] In one embodiment the cross-blocking antibodies provided by the
present invention are fully human. In one embodiment the cross-blocking
antibodies provided by the present invention are humanised. In one
embodiment the cross-blocking antibodies provided by the present
invention have an affinity for human IL-13 of 100 pM or better. In one
embodiment the cross-blocking antibodies provided by the present
invention have an affinity for human IL-13 of 50 pM or better.

[0147] In one embodiment the cross-blocking antibody has an isoelectric
point of at least 7, for example at least 8, such as 8.5, 8.6, 8.7, 8.8,
8.9 or 9.0.

[0148] The antibody molecules of the present invention suitably have a
high binding affinity, in particular picomolar affinity. Affinity may be
measured using any suitable method known in the art, including by surface
Plasmon resonance, including BIAcore as described in the Examples herein
using isolated natural or recombinant IL-13. In one example affinity is
measured using recombinant human IL-13 as described in the Examples
herein. In one embodiment the antibody molecule of the present invention
has a binding affinity of about 100 pM or better. In one embodiment the
antibody molecule of the present invention has a binding affinity of
about 50 pM or better. In one embodiment the antibody molecule of the
present invention has a binding affinity of about 40 pM or better. In one
embodiment the antibody molecule of the present invention has a binding
affinity of about 30 pM or better. In one embodiment the antibody
molecule of the present invention has a binding affinity of about 20 pM
or better. In one embodiment the antibody molecule of the present
invention is fully human or humanised and has a binding affinity of about
100 pM or better. In one embodiment the antibody molecule of the present
invention is fully human or humanised and has a binding affinity of 30 pM
or better.

[0150] In one embodiment the antibody molecules of the present invention
block the interaction between IL-13 and an IL-13 receptor, in particular
the antibody molecules of the present invention block the interaction
between IL-13 and IL-13Rα1 and the interaction between IL-13 and
IL-13Rα2. Numerous assays suitable for determining the ability of
an antibody to block this interaction are described in the examples
herein. In one embodiment the present invention provides a neutralising
antibody having specificity for human IL-13. In one embodiment the human
IL-13 receptor used in the assay is natural human IL-13Rα1 or
natural human IL-13Rα2. In one embodiment the human IL-13 receptor
used in the assay is recombinant human IL-13Rα1 or recombinant
human IL-13Rα2. In one embodiment the human IL-13 used in the assay
is recombinant human IL-13. In one embodiment the neutralising antibody
is a humanised or fully human antibody or fragment thereof.

[0151] If desired an antibody for use in the present invention may be
conjugated to one or more effector molecule(s). It will be appreciated
that the effector molecule may comprise a single effector molecule or two
or more such molecules so linked as to form a single moiety that can be
attached to the antibodies of the present invention. Where it is desired
to obtain an antibody fragment linked to an effector molecule, this may
be prepared by standard chemical or recombinant DNA procedures in which
the antibody fragment is linked either directly or via a coupling agent
to the effector molecule. Techniques for conjugating such effector
molecules to antibodies are well known in the art (see, Hellstrom et al.,
Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp.
623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et
al., 1999, Pharmacology and Therapeutics, 83, 67-123). Particular
chemical procedures include, for example, those described in WO 93/06231,
WO 92/22583, WO 89/00195, WO 89/01476 and WO 03031581. Alternatively,
where the effector molecule is a protein or polypeptide the linkage may
be achieved using recombinant DNA procedures, for example as described in
WO 86/01533 and EP 0392745.

[0155] Other effector molecules may include chelated radionuclides such as
111In and 90Y, Lu177, Bismuth213,
Californium252, Iridium192 and
Tungsten188/Rhenium188; or drugs such as but not limited to,
alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

[0158] In another example the effector molecule may increase the half-life
of the antibody in vivo, and/or reduce immunogenicity of the antibody
and/or enhance the delivery of an antibody across an epithelial barrier
to the immune system. Examples of suitable effector molecules of this
type include polymers, albumin, albumin binding proteins or albumin
binding compounds such as those described in WO 05/117984.

[0159] Where the effector molecule is a polymer it may, in general, be a
synthetic or a naturally occurring polymer, for example an optionally
substituted straight or branched chain polyalkylene, polyalkenylene or
polyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g.
a homo- or hetero-polysaccharide.

[0160] Specific optional substituents which may be present on the
above-mentioned synthetic polymers include one or more hydroxy, methyl or
methoxy groups.

[0163] "Derivatives" as used herein is intended to include reactive
derivatives, for example thiol-selective reactive groups such as
maleimides and the like. The reactive group may be linked directly or
through a linker segment to the polymer. It will be appreciated that the
residue of such a group will in some instances form part of the product
as the linking group between the antibody fragment and the polymer.

[0164] The size of the polymer may be varied as desired, but will
generally be in an average molecular weight range from 500 Da to 50000
Da, for example from 5000 to 40000 Da such as from 20000 to 40000 Da. The
polymer size may in particular be selected on the basis of the intended
use of the product for example ability to localize to certain tissues
such as tumors or extend circulating half-life (for review see Chapman,
2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example,
where the product is intended to leave the circulation and penetrate
tissue it may be advantageous to use a small molecular weight polymer,
for example with a molecular weight of around 5000 Da. For applications
where the product remains in the circulation, it may be advantageous to
use a higher molecular weight polymer, for example having a molecular
weight in the range from 20000 Da to 40000 Da.

[0165] Suitable polymers include a polyalkylene polymer, such as a
poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or a
derivative thereof, and especially with a molecular weight in the range
from about 15000 Da to about 40000 Da.

[0166] In one example antibodies for use in the present invention are
attached to poly(ethyleneglycol) (PEG) moieties. In one particular
example the antibody is an antibody fragment and the PEG molecules may be
attached through any available amino acid side-chain or terminal amino
acid functional group located in the antibody fragment, for example any
free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids
may occur naturally in the antibody fragment or may be engineered into
the fragment using recombinant DNA methods (see for example U.S. Pat. No.
5,219,996; U.S. Pat. No. 5,667,425; WO 98/25971). In one example the
antibody molecule of the present invention is a modified Fab fragment
wherein the modification is the addition to the C-terminal end of its
heavy chain one or more amino acids to allow the attachment of an
effector molecule. Suitably, the additional amino acids form a modified
hinge region containing one or more cysteine residues to which the
effector molecule may be attached. Multiple sites can be used to attach
two or more PEG molecules.

[0167] Suitably PEG molecules may be covalently linked through a thiol
group of at least one cysteine residue located in the antibody fragment.
Each polymer molecule attached to the modified antibody fragment may be
covalently linked to the sulphur atom of a cysteine residue located in
the fragment. The covalent linkage will generally be a disulphide bond
or, in particular, a sulphur-carbon bond. Where a thiol group is used as
the point of attachment appropriately activated effector molecules, for
example thiol selective derivatives such as maleimides and cysteine
derivatives may be used. An activated polymer may be used as the starting
material in the preparation of polymer-modified antibody fragments as
described above. The activated polymer may be any polymer containing a
thiol reactive group such as an α-halocarboxylic acid or ester,
e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone or a
disulphide. Such starting materials may be obtained commercially (for
example from Nektar, formerly Shearwater Polymers Inc., Huntsville, Ala.,
USA) or may be prepared from commercially available starting materials
using conventional chemical procedures. Particular PEG molecules include
20K methoxy-PEG-amine (obtainable from Nektar, formerly Shearwater; Rapp
Polymere; and SunBio) and M-PEG-SPA (obtainable from Nektar, formerly
Shearwater).

[0168] In one embodiment, the antibody is a modified Fab fragment or diFab
which is PEGylated, i.e. has PEG (poly(ethyleneglycol)) covalently
attached thereto, e.g. according to the method disclosed in EP 0948544 or
EP 1090037 [see also "Poly(ethyleneglycol) Chemistry, Biotechnical and
Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New
York, "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997,
J. Milton Harris and S. Zalipsky (eds), American Chemical Society,
Washington DC and "Bioconjugation Protein Coupling Techniques for the
Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New
York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-5451.
In one example PEG is attached to a cysteine in the hinge region. In one
example, a PEG modified Fab fragment has a maleimide group covalently
linked to a single thiol group in a modified hinge region. A lysine
residue may be covalently linked to the maleimide group and to each of
the amine groups on the lysine residue may be attached a
methoxypoly(ethyleneglycol) polymer having a molecular weight of
approximately 20,000 Da. The total molecular weight of the PEG attached
to the Fab fragment may therefore be approximately 40,000 Da.

[0169] In one embodiment, the present invention provides an antagonistic
antibody molecule having specificity for human IL-13, which is a modified
Fab' fragment having a heavy chain comprising the sequence given in SEQ
ID NO:35 and a light chain comprising the sequence given in SEQ ID NO:27
and having at the C-terminal end of its heavy chain a modified hinge
region containing at least one cysteine residue to which an effector
molecule is attached. Suitably the effector molecule is PEG and is
attached using the methods described in (WO 98/25971 and WO 2004072116 or
in WO 2007/003898. Effector molecules may be attached to antibody
fragments using the methods described in International patent
applications WO 2005/003169, WO 2005/003170 and WO 2005/003171.

[0170] In one embodiment the antibody or fragment is not attached an
effector molecule.

[0171] The present invention also provides an isolated DNA sequence
encoding the heavy and/or light chain(s) of an antibody molecule of the
present invention. Suitably, the DNA sequence encodes the heavy or the
light chain of an antibody molecule of the present invention. The DNA
sequence of the present invention may comprise synthetic DNA, for
instance produced by chemical processing, cDNA, genomic DNA or any
combination thereof.

[0172] DNA sequences which encode an antibody molecule of the present
invention can be obtained by methods well known to those skilled in the
art. For example, DNA sequences coding for part or all of the antibody
heavy and light chains may be synthesised as desired from the determined
DNA sequences or on the basis of the corresponding amino acid sequences.

[0173] DNA coding for acceptor framework sequences is widely available to
those skilled in the art and can be readily synthesised on the basis of
their known amino acid sequences.

[0174] Standard techniques of molecular biology may be used to prepare DNA
sequences coding for the antibody molecule of the present invention.
Desired DNA sequences may be synthesised completely or in part using
oligonucleotide synthesis techniques. Site-directed mutagenesis and
polymerase chain reaction (PCR) techniques may be used as appropriate.

[0175] Examples of suitable sequences are provided herein. A suitable
signal peptide for the heavy chain may be encoded therein such as the
murine signal peptide MEWSWVFLFF LSVTTGVHS (SEQ ID NO: 45). A suitable
signal peptide for the light chain may be encoded therein such as the
murine signal peptide MSVPTQVLGL LLLWLTDARC (SEQ ID NO: 46) which is
cleaved to give an antagonistic antibody molecule of the present
invention. The present invention also provides an isolated DNA sequence
encoding the heavy chain of an antibody of the present invention which
comprises SEQ ID NO: 32, 34 or 36 or 38. The present invention also
provides an isolated DNA sequence encoding the light chain of an antibody
of the present invention which comprises SEQ ID NO:24, 26, 28 or 30.

[0176] General methods by which the vectors may be constructed,
transfection methods and culture methods are well known to those skilled
in the art. In this respect, reference is made to "Current Protocols in
Molecular Biology", 1999, F. M. Ausubel (ed), Wiley Interscience, New
York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0177] Also provided is a host cell comprising one or more cloning or
expression vectors comprising one or more DNA sequences encoding an
antibody of the present invention. Any suitable host cell/vector system
may be used for expression of the DNA sequences encoding the antibody
molecule of the present invention. Bacterial, for example E. coli, and
other microbial systems may be used or eukaryotic, for example mammalian,
host cell expression systems may also be used. Suitable mammalian host
cells include CHO, myeloma or hybridoma cells.

[0178] The present invention also provides a process for the production of
an antibody molecule according to the present invention comprising
culturing a host cell containing a vector of the present invention under
conditions suitable for leading to expression of protein from DNA
encoding the antibody molecule of the present invention, and isolating
the antibody molecule.

[0179] The antibody molecule may comprise only a heavy or light chain
polypeptide, in which case only a heavy chain or light chain polypeptide
coding sequence needs to be used to transfect the host cells. For
production of products comprising both heavy and light chains, the cell
line may be transfected with two vectors, a first vector encoding a light
chain polypeptide and a second vector encoding a heavy chain polypeptide.
Alternatively, a single vector may be used, the vector including
sequences encoding light chain and heavy chain polypeptides.

[0180] The antibodies and fragments according to the present disclosure
are expressed at good levels from host cells. Thus the properties of the
antibodies and/or fragments appear to optimised and condusive to
commercial processing.

[0181] As the antibodies of the present invention are useful in the
treatment and/or prophylaxis of a pathological condition, the present
invention also provides a pharmaceutical or diagnostic composition
comprising an antibody molecule of the present invention in combination
with one or more of a pharmaceutically acceptable excipient, diluent or
carrier. Accordingly, provided is the use of an antibody of the invention
for the manufacture of a medicament. The composition will usually be
supplied as part of a sterile, pharmaceutical composition that will
normally include a pharmaceutically acceptable carrier. A pharmaceutical
composition of the present invention may additionally comprise a
pharmaceutically-acceptable adjuvant.

[0182] The present invention also provides a process for preparation of a
pharmaceutical or diagnostic composition comprising adding and mixing the
antibody molecule of the present invention together with one or more of a
pharmaceutically acceptable excipient, diluent or carrier.

[0183] The antibody molecule may be the sole active ingredient in the
pharmaceutical or diagnostic composition or may be accompanied by other
active ingredients including other antibody ingredients, for example
anti-TNF, anti-IL-1β, anti-T cell, anti-IFNγ or anti-LPS
antibodies, or non-antibody ingredients such as xanthines. Other suitable
active ingredients include antibodies capable of inducing tolerance, for
example, anti-CD3 or anti-CD4 antibodies.

[0184] In a further embodiment the antibody, fragment or composition
according to the disclosure is employed in combination with a further
pharmaceutically active agent, for example a corticosteroid (such as
fluticasonoe propionate) and/or a beta-2-agonist (such as salbutamol,
salmeterol or formoterol) or inhibitors of cell growth and proliferation
(such as rapamycin, cyclophosphmide, methotrexate) or alternatively a
CD28 and/or CD40 inhibitor. In one embodiment the inhibitor is a small
molecule. In another embodiment the inhibitor is an antibody specific to
the target.

[0185] The pharmaceutical compositions suitably comprise a therapeutically
effective amount of the antibody of the invention. The term
"therapeutically effective amount" as used herein refers to an amount of
a therapeutic agent needed to treat, ameliorate or prevent a targeted
disease or condition, or to exhibit a detectable therapeutic or
preventative effect. For any antibody, the therapeutically effective
amount can be estimated initially either in cell culture assays or in
animal models, usually in rodents, rabbits, dogs, pigs or primates. The
animal model may also be used to determine the appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and routes for administration in humans.

[0186] The precise therapeutically effective amount for a human subject
will depend upon the severity of the disease state, the general health of
the subject, the age, weight and gender of the subject, diet, time and
frequency of administration, drug combination(s), reaction sensitivities
and tolerance/response to therapy. This amount can be determined by
routine experimentation and is within the judgement of the clinician.
Generally, a therapeutically effective amount will be from 0.01 mg/kg to
50 mg/kg, for example 0.1 mg/kg to 20 mg/kg. Alternatively, the dose may
be 1 to 500 mg per day such as 10 to 100, 200, 300 or 400 mg per day.
Pharmaceutical compositions may be conveniently presented in unit dose
forms containing a predetermined amount of an active agent of the
invention.

[0187] Compositions may be administered individually to a patient or may
be administered in combination (e.g. simultaneously, sequentially or
separately) with other agents, drugs or hormones.

[0188] The dose at which the antibody molecule of the present invention is
administered depends on the nature of the condition to be treated, the
extent of the inflammation present and on whether the antibody molecule
is being used prophylactically or to treat an existing condition.

[0189] The frequency of dose will depend on the half-life of the antibody
molecule and the duration of its effect. If the antibody molecule has a
short half-life (e.g. 2 to 10 hours) it may be necessary to give one or
more doses per day. Alternatively, if the antibody molecule has a long
half life (e.g. 2 to 15 days) it may only be necessary to give a dosage
once per day, once per week or even once every 1 or 2 months.

[0190] The pharmaceutically acceptable carrier should not itself induce
the production of antibodies harmful to the individual receiving the
composition and should not be toxic. Suitable carriers may be large,
slowly metabolised macromolecules such as proteins, polypeptides,
liposomes, polysaccharides, polylactic acids, polyglycolic acids,
polymeric amino acids, amino acid copolymers and inactive virus
particles.

[0191] Pharmaceutically acceptable salts can be used, for example mineral
acid salts, such as hydrochlorides, hydrobromides, phosphates and
sulphates, or salts of organic acids, such as acetates, propionates,
malonates and benzoates.

[0192] Pharmaceutically acceptable carriers in therapeutic compositions
may additionally contain liquids such as water, saline, glycerol and
ethanol. Additionally, auxiliary substances, such as wetting or
emulsifying agents or pH buffering substances, may be present in such
compositions. Such carriers enable the pharmaceutical compositions to be
formulated as tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries and suspensions, for ingestion by the patient.

[0193] Suitable forms for administration include forms suitable for
parenteral administration, e.g. by injection or infusion, for example by
bolus injection or continuous infusion. Where the product is for
injection or infusion, it may take the form of a suspension, solution or
emulsion in an oily or aqueous vehicle and it may contain formulatory
agents, such as suspending, preservative, stabilising and/or dispersing
agents. Alternatively, the antibody molecule may be in dry form, for
reconstitution before use with an appropriate sterile liquid.

[0194] Once formulated, the compositions of the invention can be
administered directly to the subject. The subjects to be treated can be
animals. However, in one or more embodiments the compositions are adapted
for administration to human subjects.

[0195] In one embodiment, in formulations according to the present
disclosure, the pH of the final formulation is not similar to the value
of the isoelectric point of the antibody or fragment, for if the pH of
the formulation is 7 then a pI of from 8-9 or above may be appropriate.
Whilst not wishing to be bound by theory it is thought that this may
ultimately provide a final formulation with improved stability, for
example the antibody or fragment remains in solution.

[0196] The pharmaceutical compositions of this invention may be
administered by any number of routes including, but not limited to, oral,
intravenous, intramuscular, intra-arterial, intramedullary, intrathecal,
intraventricular, transdermal, transcutaneous (for example, see
WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, intravaginal or rectal routes. Hyposprays may also be used to
administer the pharmaceutical compositions of the invention. Typically,
the therapeutic compositions may be prepared as injectables, either as
liquid solutions or suspensions. Solid forms suitable for solution in, or
suspension in, liquid vehicles prior to injection may also be prepared.
Preferably the antibody molecules of the present invention are
administered subcutaneously, by inhalation or topically.

[0197] Direct delivery of the compositions will generally be accomplished
by injection, subcutaneously, intraperitoneally, intravenously or
intramuscularly, or delivered to the interstitial space of a tissue. The
compositions can also be administered into a specific tissue of interest.
Dosage treatment may be a single dose schedule or a multiple dose
schedule.

[0198] It will be appreciated that the active ingredient in the
composition will be an antibody molecule. As such, it will be susceptible
to degradation in the gastrointestinal tract. Thus, if the composition is
to be administered by a route using the gastrointestinal tract, the
composition will need to contain agents which protect the antibody from
degradation but which release the antibody once it has been absorbed from
the gastrointestinal tract.

[0200] In one embodiment the formulation is provided as a formulation for
topical administrations including inhalation.

[0201] Suitable inhalable preparations include inhalable powders, metering
aerosols containing propellant gases or inhalable solutions free from
propellant gases (such as nebulisable solutions or suspensions).
Inhalable powders according to the disclosure containing the active
substance may consist solely of the abovementioned active substances or
of a mixture of the above mentioned active substances with
physiologically acceptable excipient.

[0202] These inhalable powders may include monosaccharides (e.g. glucose
or arabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo-
and polysaccharides (e.g. dextrans), polyalcohols (e.g. sorbitol,
mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or
mixtures of these with one another. Mono- or disaccharides are suitably
used, the use of lactose or glucose, particularly but not exclusively in
the form of their hydrates.

[0203] Particles for deposition in the lung require a particle size less
than 10 microns, such as 1-9 microns for example from 0.1 to 5 μm, in
particular from 1 to 5 μm. The particle size of the active (such as
the antibody or fragment is of primary importance).

[0204] The propellent gases which can be used to prepare the inhalable
aerosols are known in the art. Suitable propellent gases are selected
from among hydrocarbons such as n-propane, n-butane or isobutane and
halohydrocarbons such as chlorinated and/or fluorinated derivatives of
methane, ethane, propane, butane, cyclopropane or cyclobutane. The
abovementioned propellent gases may be used on their own or in mixtures
thereof.

[0206] The propellent-gas-containing inhalable aerosols may also contain
other ingredients such as cosolvents, stabilisers, surface-active agents
(surfactants), antioxidants, lubricants and means for adjusting the pH.
All these ingredients are known in the art.

[0207] The propellant-gas-containing inhalable aerosols according to the
invention may contain up to 5% by weight of active substance. Aerosols
according to the invention contain, for example, 0.002 to 5% by weight,
0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% by weight, 0.5 to
2% by weight or 0.5 to 1% by weight of active.

[0208] Alternatively topical administrations to the lung may also be by
administration of a liquid solution or suspension formulation, for
example employing a device such as a nebulizer, for example, a nebulizer
connected to a compressor (e.g., the Pari LC-Jet Plus® nebulizer
connected to a Pari Master® compressor manufactured by Pari
Respiratory Equipment, Inc., Richmond, Va.).

[0209] In one embodiment the formulation is provided as discrete ampoules
containing a unit dose for delivery by nebulisation.

[0210] In one embodiment the antibody is supplied in lyophilised form, for
reconstitutions or alternatively as a suspension formulation.

[0211] The antibody of the invention can be delivered dispersed in a
solvent, e.g., in the form of a solution or a suspension. It can be
suspended in an appropriate physiological solution, e.g., physiological
saline, a pharmacologically acceptable solvent or a buffered solution.
Buffered solutions known in the art may contain 0.05 mg to 0.15 mg
disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate,
0.25 mg to 0.30 mg anhydrous citric acid, and 0.45 mg to 0.55 mg sodium
citrate per 1 ml of water so as to achieve a pH of about 4.0 to 5.0. As
mentioned supra a suspension can made, for example, from lyophilised
antibody.

[0212] The therapeutic suspensions or solution formulations can also
contain one or more excipients. Excipients are well known in the art and
include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer
and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,
phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride,
liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions can
be encapsulated in liposomes or biodegradable microspheres. The
formulation will generally be provided in a substantially sterile form
employing sterile manufacture processes.

[0213] This may include production and sterilization by filtration of the
buffered solvent solution used for the formulation, aseptic suspension of
the antibody in the sterile buffered solvent solution, and dispensing of
the formulation into sterile receptacles by methods familiar to those of
ordinary skill in the art.

[0214] Nebulisable formulation according to the present disclosure may be
provided, for example, as single dose units (e.g., sealed plastic
containers or vials) packed in foil envelopes. Each vial contains a unit
dose in a volume, e.g., 2 ml, of solvent/solution buffer.

[0215] The antibodies of the present disclosure are thought to be suitable
for delivery via nebulisation.

[0216] It is also envisaged that the antibody of the present invention may
be administered by use of gene therapy. In order to achieve this, DNA
sequences encoding the heavy and light chains of the antibody molecule
under the control of appropriate DNA components are introduced into a
patient such that the antibody chains are expressed from the DNA
sequences and assembled in situ.

[0217] The present invention also provides an antibody molecule (or
compositions comprising same) for use in the control of inflammatory
diseases, for example acute or chronic inflammatory disease. Suitably,
the antibody molecule (or compositions comprising same) can be used to
reduce the inflammatory process or to prevent the inflammatory process.
In one embodiment there is provided an in vivo reduction of activated T
cells, in particular those involved in inappropriate inflammatory immune
responses, for example recruited to the vicinity/location of such a
response.

[0218] Reduction of activated T cells, as employed herein, may be a
reduction, 10, 20, 30, 40, 50, 60, 70, 80, 90 or more percent in
comparison before treatment or without treatment.

[0219] Advantageously, treatment with an antibody, fragment or composition
according to the present invention, may allow the reduction in the level
of activated T cells, without reducing the patients general level of T
cells (unactivated T cells). This may result in fewer side effects, and
possibly prevent T cell depletion in the patient.

[0220] The present invention also provides the antibody molecule of the
present invention for use in the treatment or prophylaxis of a
pathological disorder that is mediated by IL-13 or associated with an
increased level of IL-13.

[0222] The present invention also provides an antibody molecule according
to the present invention for use in the treatment or prophylaxis of pain,
particularly pain associated with inflammation.

[0223] In one embodiment the antibody according to the invention reduces
resistance to treatment of inflammation, particularly lung resistance to
treatment of inflammation.

[0224] In one embodiment the antibody according to the invention reduces
IL-13 protein levels in brochial tissue, for example in comparison to the
levels before treatment. The reduction may be 5, 10, 20, 30, 40% or more.

[0225] In one embodiment the antibody according to the invention reduces
IL-13 protein levels in nasal lavage fluid and/or bronchoalveolar fluid,
for example in comparison to the levels before treatment. The reduction
may be 5, 10, 20, 30, 40% or more.

[0226] In one embodiment the antibody according to the invention reduces
eosinophil influx, for example in comparison to the levels before
treatment. The reduction may be 5, 10, 20, 30, 40% or more, for example
when treated for 1, 2, 3, 4, 5, 6 or more weeks.

[0227] In one embodiment the antibody according to the invention is
suitable for reducing inappropriate levels of goblet cells, for example
in the treatment of goblet cell hyperplasia, such as chronic goblet cell
hyperplasia. The reduction may be observed after treatment for 1, 2, 3,
4, 5, 6 or more weeks.

[0228] In one embodiment the antibody according to the invention is
suitable for reducing the levels of exhaled nitric oxide (FeNO), in
comparison to levels before treatment. Exhaled nitric oxide is thought to
be a risk factor or marker for lung inflammation.

[0229] In one embodiment the antibody according to the invention is
suitable for prevention of inappropriate collagen deposition associated
with inflammatory responses, in particular peribronchial collagen
deposition.

[0230] In one embodiment the antibody according to the invention is
suitable for preventing inappropriate angiogenesis associated with
inflammatory responses.

[0231] Thus there is provided an antibody according to the invention for
use in treatment and methods of treatment employing same.

[0232] Antibody according to the invention as employed herein also refers
to fragments and derivatives disclosed in the specification.

[0233] The present invention further provides the use of an antibody
molecule, fragment or composition according to the present invention in
the manufacture of a medicament for the treatment or prophylaxis of a
pathological disorder that is mediated by IL-13 or associated with an
increased level of IL-13, for example as described herein, in particular
the pathological disorder is rheumatoid arthritis, asthma or COPD.

[0234] The present invention further provides the use of an antibody
molecule, fragment or composition according to the present invention in
the manufacture of a medicament for the treatment or prophylaxis of one
or more medical indications described herein.

[0235] An antibody molecule, fragment or composition of the present
invention may be utilised in any therapy where it is desired to reduce
the effects of IL-13 in the human or animal body. IL-13 may be
circulating in the body or may be present in an undesirably high level
localised at a particular site in the body, for example a site of
inflammation.

[0236] In one embodiment the antibody molecule of the present invention or
a composition comprising the same is used for the control of inflammatory
disease, for example as described herein.

[0237] The present invention also provides a method of treating human or
animal subjects suffering from or at risk of a disorder mediated by
IL-13, the method comprising administering to the subject an effective
amount of the antibody molecule of the present invention, or a
composition comprising the same. In one example the antibody molecule is
administered by inhalation.

[0238] In one example the disorder is selected from any of the medical
indications provided above. In one example the disorder is selected from
the group consisting of: asthmatic disorders, atopic disorders, chronic
obstructive pulmonary disease (COPD), conditions involving airway
inflammation, eosinophilia, fibrosis and excess mucus production,
inflammatory conditions, autoimmune conditions, tumors or cancers, viral
infection and suppression of expression of protective type 1 immune
responses.

[0239] In one embodiment there is provided a process for purifying an
antibody (in particular an antibody or fragment according to the
invention).

[0240] In one embodiment there is provided a process for purifying an
antibody (in particular an antibody or fragment according to the
invention) comprising the steps: performing anion exchange chromatography
in non-binding mode such that the impurities are retained on the column
and the antibody is maintained in the unbound fraction. The step may, for
example be performed at a pH about 6-8.

[0241] The process may further comprise an initial capture step employing
cation exchange chromatography, performed for example at a pH of about 4
to 5.

[0242] The process may further comprise of additional chromatography
step(s) to ensure product and process related impurities are
appropriately resolved from the product stream.

[0243] The purification process may also comprise of one or more
ultra-filtration steps, such as a concentration and diafiltration step.

[0244] Thus in one embodiment there is provided a purified IL-13 antibody
or fragment, for example a humanised antibody or fragment, in particular
an antibody or fragment according to the invention, in substantially
purified from, in particular free or substantially free of endotoxin
and/or host cell protein or DNA. Having said this, the antibodies
according to the present invention will generally be prepared in
mammalian cells and thus endotoxin content is not generally an issue. In
fact endotoxin content is more a consideration when the antibodies are
prepared in bacterial cells.

[0245] Purified form as used supra is intended to refer to at least 90%
purity, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w or more pure.

[0246] Substantially free of endotoxin is generally intended to refer to
an endotoxin content of 1 EU per mg antibody product or less such as 0.5
or 0.1 EU per mg product.

[0247] Substantially free of host cell protein or DNA is generally
intended to refer to host cell protein and/or DNA content 400 μg per
mg of antibody product or less such as 100 μg per mg or less, in
particular 20 μg per mg, as appropriate.

[0248] The antibody molecule of the present invention may also be used in
diagnosis, for example in the in vivo diagnosis and imaging of disease
states involving IL-13.

[0249] Suitable in vivo assays for testing the properties of the
antibodies according to the invention include: the chronic house mite
model, hyperresponsiveness to methacholine and/or the ovalbumin model of
allergic lung inflammation.

[0250] Comprising in the context of the present specification is intended
to meaning including.

[0251] Where technically appropriate embodiments of the invention may be
combined.

[0252] Embodiments are described herein as comprising certain
features/elements. The disclosure also extends to separate embodiments
consisting or consisting essentially of said features/elements.

[0253] The present invention is further described by way of illustration
only in the following examples, which refer to the accompanying Figures,
in which:

[0300] shows an alignment of the light chains for the
rat, acceptor framework and the humanised light chains and also heavy
chains. CDRs are in bold and underlined. Donor residues G49 and R71 are
in bold, italic and highlighted.

[0301] FIG. 13 Effect of Ab652 on BAL eotaxin-3 measured 24 h after
allergen challenge in a non-human primate model of asthma. Data are
expressed as mean±SEM, n=4-8 per group.

[0302] FIG. 14. Effect of Ab652 on the BAL eosinophil count measured 24 h
after allergen challenge in a non-human primate model of asthma. Data are
normalised to the BAL eosinophil count measured in the screening phase of
the study. Mean±SEM, n=4-8 per group.

[0303] FIG. 15. Effect of Ab652 on peak airway resistance measured up to
15 minutes after allergen challenge in a non-human primate model of
asthma. Data are expressed as mean±SEM, n=4-8 per group.

[0305] Rats were immunised with either purified human IL-13 (Peprotech) or
rat fibroblasts expressing human IL-13 (expressing approx 1 ug/ml in
culture supernatant), or in some cases, a combination of the two.
Following 3 to 6 shots, animals were sacrificed and PBMC, spleen, bone
marrow and lymph nodes harvested. Sera was monitored for binding to human
IL-13 in ELISA and also for the ability to neutralise hIL-13 in the
HEK-293 STAT-6 reporter cell assay (HEK-Blue assay, Invivogen).

[0307] SLAM culture supernatants were first screened for their ability to
bind hIL-13 in a bead-based assay in the FMAT. This was a homogeneous
assay using biotinylated human IL-13 coated onto streptavidin beads and a
goat anti-rat Fc-Cy5 conjugate. Positives from this assay were then fed
into the HEK-293 IL-13R-STAT-6 reporter cell assay (HEK-Blue assay,
Invivogen) to identify neutralisers. Neutralising supernatants were then
profiled in the Biacore to estimate off-rate and also to characterise the
mode of action of neutralisation. Neutralisation was categorised as
either bin 1 or bin 2. Bin 1 represented an antibody that binds to human
IL-13 and prevents binding of IL-13Rα1 and as a result also blocks
IL-4R from binding. Bin 2 represented an antibody that binds hIL-13 in
such a way that allows binding to IL-13Rα1 but prevents recruitment
of IL-4R into the complex. We were selecting antibodies that operated via
bin 1.

[0309] Humanisation of all 5 families was performed. Based on
neutralisation potency, affinity and donor content in humanised grafts,
humanised CA154--652 (see below) was selected for further
progression.

1.1 Humanisation

[0310] The humanised antibody exemplified herein (Ab652) was prepared by
grafting the CDRs from the rat antibody V-regions (Seq ID NOs 7 and 15)
(CDRs disclosed herein in sequences 1 to 6) onto human germline antibody
V-region frameworks. Alignments of the rat antibody (donor) V-region
sequences with the human germline antibody (acceptor) V-region sequences
are shown in FIG. 12, together with the designed humanised sequence. The
CDRs grafted from the donor to the acceptor sequence are as defined by
Kabat (Kabat et al. Sequence of proteins of immunological interest
(1987). Bethesda Md., National Institutes of Health, US), with the
exception of CDR-H1 where the combined Chothia/Kabat definition is used
(see Adair et al. (1991) Humanised antibodies WO 91/09967). Human
V-region VH2 3-12-26 plus JH4 J-region (V BASE,
http://vbase.mrc-cpe.cam.ac.uk/) was chosen as the acceptor for the heavy
chain CDRs. The heavy chain framework residues are all from the human
germline gene, with the exception of residues 49 and 71 (Kabat
numbering), where the donor residues Glycine (G49) and Arginine (R71)
were retained, respectively. Retention of these two donor residues was
essential for full activity of the humanised antibody. Human V-region VK1
2-1-(1) 02 plus JK4 J-region (V BASE, http://vbase.mrc-cpe.cam.ac.uk/)
was chosen as the acceptor for the light chain CDRs. The light chain
framework residues are all from the human germline gene.

[0311] Genes encoding initial V-region sequences were designed and
constructed by an automated synthesis approach by Entelechon GmbH. A
number of different variants of the heavy chain were created by modifying
the VH gene by oligonucleotide-directed mutagenesis. The gL1 gene
sequence was cloned into the UCB-Celltech human light chain expression
vector pKH10.1, which contains DNA encoding the human Kappa chain
constant region (Km3 allotype)--see SEQ ID No: 26 and SEQ ID NO:30. The
eight grafted VH genes (gH1 to gH8) were cloned into the UCB-Celltech
human gamma-4 heavy chain expression vector pVhγ4P FL, which
contains DNA encoding the human gamma-4 heavy chain constant region with
the hinge stabilising mutation S241P (Angal et al., Mol Immunol. 1993,
30(1):105-8). The gH2 VH gene was selected as the optimum heavy chain
graft for potency and biophysical characteristics (described herein
below), and was then sub-cloned into the UCB-Celltech human gamma-1 Fab
vector pVhγ1F3, which contains DNA encoding the human gamma-1 CH1
domain (G1m17 allotype) (SEQ ID NO:38). Co-transfection of the resulting
heavy chain plasmid with the light chain plasmid, into CHO-L761h cells
resulted in the expression of the humanised antibody in the required Fab
format. This antibody is referred to herein as Ab652 (also referred to as
Ab652 Fab).

[0312] To facilitate the generation of a stable cell line expressing
antibody Ab652, a single plasmid containing DNA encoding both the heavy
and light chain expression cassettes and a glutamine synthetase (GS)
selection marker was generated. The GS gene allows for the selection of
recombinant CHO cells by permitting growth in media supplemented with the
GS inhibitor methionine sulphoximine (Bebbington et al., Biotechnol.
1992, 10(2): 169-175).

1.2 Binding Affinity Measurements

[0313] The BIAcore technology monitors the binding between biomolecules in
real time and without the requirement for labelling. One of the
interactants, termed the ligand, is either immobilised directly or
captured on the immobilised surface while the other, termed the analyte,
flows in solution over the captured surface. The sensor detects the
change in mass on the sensor surface as the analyte binds to the ligand
to form a complex on the surface. This corresponds to the association
process. The dissociation of the analyte from the ligand is monitored
when the analyte is replaced by buffer. In the affinity BIAcore assay,
the ligand is Ab652 and the analyte is human IL-13.

1.3 Receptor Cross-Blocking Assay

[0314] The Biacore receptor cross-blocking assay requires the capture of
anti IL-13 Fab followed by IL-13 (as the first analyte) flowed over the
captured ligand to form a stable complex on the sensor surface. A second
analyte (recombinant soluble IL-13 receptor) is then flowed over this
stable complex. The amount of binding of the second analyte to the stable
complex is monitored. Anti IL-13 antibodies that do not allow the second
analyte to bind to the stable antibody:IL-13 complex are classified as
Site 1 competitors. Those anti IL-13 antibodies that allow the second
analyte to bind to the stable antibody:IL-13 complex are classified as
Site 2 competitors.

[0316] Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).
Made up to 75 mg/mL in distilled water and stored in 200 μL aliquots
at -70° C. N-Hydroxysuccinimide (NHS). Made up to 11.5 mg/mL in
distilled water and stored in 200 μL aliquots at -70° C. 1 M
Ethanolamine hydrochloride-NaOH pH 8.5. Stored in 200 μL aliquots at
-70° C.

[0323] The assay format was capture of the Ab652 by immobilised anti-human
F(ab')2 then titration of the human hIL-13 over the captured
surface.

[0324] BIA (Biamolecular Interaction Analysis) was performed using a
BIAcore 3000 (BIAcore AB). Affinipure F(ab')2 Fragment, goat
anti-human IgG, F(ab')2 fragment specific (Jackson ImmunoResearch)
was immobilised on a CM5 Sensor Chip via amine coupling chemistry to a
capture level of ≈4000 response units (RUs). A blank surface was
prepared in a similar way, omitting the F(ab')2 fragment from the
procedure. HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,
0.005% Surfactant P20, BIAcore AB) was used as the running buffer with a
flow rate of 10 μl/min A 10 μl injection of Ab652 Fab at ˜0.2
μg/mL was used for capture by the immobilised anti-human
IgG-F(ab')2 to allow sufficient IL-13 binding but also to minimise
mass transport limited binding effects. Human IL13 was titrated over the
captured Ab652 at various concentrations (10 nM to 0.31 nM) at a flow
rate of 30 μL/min. The surface was regenerated by a 10 μL injection
of 40 mM HCl, followed by a 5 μL injection of mM NaOH at a flowrate of
10 μL/min

[0326] The BIAcore receptor cross-blocking assay requires the capture of
anti-IL-13 Fab followed by IL-13 (as the first analyte) flowed over the
capture ligand to form a stable complex on the sensor surface. A second
analyte (recombinant soluble IL-13 receptor) is then flowed over this
stable complex. The amount of binding of the second analyte to the stable
complex is then monitored. Anti-IL-13 antibodies that do not allow the
second analyte to bind to the stable antibody:IL-13 complex are
classified as Axis 1 competitors. Those anti IL-13 antibodies that allow
the second analyte to bind the stable antibody:IL-13 complex are
classified as Axis 2 competitors.

[0327] All experiments were performed using at Biacore 3000 biosensor at
25° C. HBS-EP buffer was used as the running buffer with a flow
rate of 10 μL/minute (min). The same sensor surfaces were used as
described for the affinity determinations.

[0328] A 10 μl injection of ˜0.2 μg/ml of the anti human IL-13
Fab was used for capture by the goat F(ab')2 IgG, anti-human
F(ab')2-fragment specific sensor surface. The anti human IL-13 Fab at 0.2
μg/ml gave sufficient anti-human IL-13 binding. Human IL-13 at 25 nM
was injected over the captured antibody followed immediately by soluble
human IL-13 receptor at 100 nM at a flow rate of 10 μL/min. The
surface was regenerated by two 30 μL injections of 40 mM HCl, followed
by a 5 μL injection of 5 mM NaOH at a flow rate of 30 μL/min

[0329] Background subtracted binding curves were analysed using the
BlAevaluation software provided by the manufacturer (version 3.2)
following standard procedures.

[0330] IL-13 interacts with either of two receptors (IL-13Rα1 and
IL-13Rα2) to form a complex. Only the hIL-13/hIL-13 α1
complex signals. Therefore, anti-IL13 antibodies that inhibit IL-13
dependent signalling can mediate this effect by blocking the interaction
with hIL-13 α1. The site of interaction of Ab652 on human IL-13 was
to be determined in a BIAcore assay. Ab652 was captured by an immobilised
anti-human F(ab')2 surface and then hIL-13 was in turn captured by
the Ab652. The binding of the soluble IL-13Rα1 to the captured
IL-13/antibody complex was assessed. The assay was repeated with
hIL-13Rα2 substituted for hIL-13Rα1. IL-13 presented by Ab652
could not bind either of the IL-13 receptors, but a commercial control
anti-IL-13 antibody (mAb 213) was capable of presenting IL-13 to the
soluble IL-13 receptor. In conclusion, Ab652 inhibits IL-13 binding to
both the hIL-13 receptor subunits, defining it as an Axis 1 competitor.

The affinity of Ab652 for hIL-13 was determined in a Biacore assay over
three separate assays. The affinity was in the range 9-24 pM with a mean
of 13.4 (+4.8) pM

1.6 Cell-Based Potency

[0331] The in vitro potency of Ab652 Fab to neutralise IL-13 was
investigated using the HEK-BLUE® STAT-6 assay (Invivogen). The assay
comprises HEK293 cells stably expressing human STAT-6 and stably
expressing secreted embryonic alkaline phosphatase (SEAP) under the
control of the IFN-β minimal promoter fused to four STAT-6 binding
sites. The neutralisation potency (IC50) of Ab652 was assessed using
different types of human IL-13, used in the assay at 250 pg/mL.
Neutralisation potency was assessed against recombinant wild-type human
IL-13 produced from bacterial (E. coli) and mammalian (rat fibroblasts)
host cells. Neutralisation potency was assessed against natural wild-type
and variant R130Q human IL-13 produced from human T-lymphocytes and
against recombinant cynomolgus monkey IL-13 produced in mammalian cells.
R130Q hIL-13 was not purified and the concentration was determined by
hIL-13 ELISA. Cynomolgus IL-13 was not purified and was used at a
concentration giving an equivalent response in the assay as 250 pg/mL MIA
3. In addition, the neutralisation potency of CA154--652.g2 Fab was
measured following nebulisation using the PARI eFLOW® mesh nebuliser.
Table 4: IC50 values of Ab652 Fab against multiple forms of IL-13 in
the HEK Blue Assay. For determination of functional affinity IL-13
titrations were performed in the presence of fixed concentrations of
Ab652. Schild-plot analysis was applied to data to determine KD
values for neutralisation of recombinant human wild-type IL-13 and
recombinant cynomolgus monkey IL-13. Table 5: IC50 and KD
values of Ab652 Fab against multiple forms of IL-13 in the HEK Blue
Assay.

[0332] Overall, these data demonstrate that Ab652 Fab is similarly potent
at neutralising recombinant and natural human IL-13 produced from
bacterial and mammalian sources. The potency of CA154--652.g2
against cynomolgus IL-13 in this assay is no more than 3-fold lower than
against human IL-13 also generated from rat fibroblasts. The potency of
CA154--652.g2 is not altered following nebulisation using the PARI
eFLOW® nebuliser.

1.7 Physical Characterisation of Ab652

[0333] As described above 8 different antibody grafted variable regions
were generated using the CDRs derived from the selected rat antibody (SEQ
ID NOs: 1-6, FIG. 1). Selection of Ab652 (gL1gH2) from those 8 grafts was
based on potency as described above and biophysical characteristics.

[0334] Based on the data generated for all the grafted variable regions
tested, antibody 652 was chosen because it:

[0335] Maintained the
highest affinity against hIL-13 and variant IL-13

[0338] No aggregation on shaking or when nebulised In contrast, some
of the other grafts tested showed a reduction in binding affinity, poor
pH stability and aggregation by shaking and/or nebulisation.

1.7.1 Effect of Nebulisation

[0339] To determine whether Ab652 was suitable for nebulisation the PARI
eFLOW® nebuliser was used. Volumes of 2.5 mL of Ab652 solution in 50
mM sodium acetate/125 mM sodium chloride pH5 were nebulised at ambient
temperature (about 21° C.) and collected by condensing the
nebulisate in cooled collection tubes. Subsequent analysis indicated no
apparent degradation. The study was also repeated using a solution in
PBS, pH 7. A positive control of IgG4 was included, having been found to
aggregate during nebulisation. Analysis of the nebulised samples was by
size exclusion, SDS-PAGE, dynamic light scattering and ligand binding,
with the particular aim of detecting aggregated material present. No
change was apparent by any of those techniques indicating that Ab652 was
resistant to damage during nebulisation.

1.7.2 Summary of the Physical Characteristics of Ab652

[0340] pI (isoelectric point) 8 (average of two determinations) Thermal
stability Tm 84° C. No aggregration of Ab652 was observed when the
antibody was subjected to aggitation/shaking or nebulisation

2. Effect of Ab652 in a Non-Human Primate Model of Asthma

Objective

[0341] The objective of this study was to evaluate the efficacy of Ab652
in a non-human primate model of asthma. Primary endpoints included the
effects on bronchoalveolar lavage (BAL) cell counts, chemokine levels,
and early and late pulmonary function changes as assessed by lung
resistance (RL).

Methods

[0342] Ab652 was delivered using a mesh nebuliser. Breath simulation
studies were conducted using typical ventilator parameters and tubing
set-up used at the study facility. The results of the breath simulation
studies indicated that 40.4% of the material charge in the nebuliser
would be delivered at the level of the endotracheal tube.

[0343] Study animals were selected on the basis of historical pulmonary
function values and BAL eosinophil counts. In the screening session,
animals underwent Ascaris suum (A. suum) antigen challenge before
assignment to treatment groups in order to characterize their normal
(untreated) response to A. suum. After this screening session, animals
were assigned to dose groups on the basis of BAL cell counts and
pulmonary function data from the screening session. In the treatment
session, animals received either nebulised vehicle (PBS), or nebulised
Ab652 at dose level in the nebuliser of either 0.1, 1, 10, and 60
mg/animal/day. Doses were administered via nebuliser on Days -2, -1, 1,
2, and 3. On Days 1 and 2, treatment administration occurred
approximately 30 minutes before A. suum challenge.

[0344] Challenge procedures were identical for both sessions. Each animal
was challenged on Days 1 and 2, and pulmonary function values (RL)
were recorded for at least 15 minutes after each antigen challenge and at
24 h after each allergen challenge. BAL fluid was collected before the
first challenge and approximately 24 h after the second challenge for
evaluation of total cell numbers, morphology, and differential counts in
order to assess the degree of pulmonary inflammation. Samples of BAL
supernatant were collected and analysed for determination of chemokine
concentration.

[0346] The data generated with nebulised Ab652 in the Ascaris model of
asthma in cynomolgus monkeys demonstrate that IL-13-driven allergic lung
inflammation is sensitive to pharmacological modulation by a neutralising
anti-IL-13 Fab fragment delivered directly to the airways in an aerosol.
Significantly Ab652 was potent, demonstrating efficacy at low mg/day
doses.

[0347] It will of course be understood that the present invention has been
described by way of example only, is in no way meant to be limiting, and
that modifications of detail can be made within the scope of the claims
hereinafter. Preferred features of each embodiment of the invention are
as for each of the other embodiments mutatis mutandis. All publications,
including but not limited to patents and patent applications, cited in
this specification are herein incorporated by reference as if each
individual publication were specifically and individually indicated to be
incorporated by reference herein as though fully set forth.